rna-isolation-purification-cells-primary-rat-brain-microvascular-endothelial-cells

Get tips on using Gibco™ RPMI 1640 Medium to perform Stem cell culture media Mouse trophoblast stem cells

Get tips on using Gibco™DMEM/F-12 to perform Stem cell culture media Mouse trophoblast stem cells

Get tips on using DeadEnd™ Colorimetric TUNEL System to perform TUNEL assay cell type - Mouse skeletal muscle cells

Get tips on using MouseTRAP™ (TRAcP 5b) ELISA to perform Acid phosphatase assay cell type - murine macrophage cells

Get tips on using ICAfectin®442 siRNA transfection to perform DNA transfection Mammalian cells - Immortalized cell lines COS7

Get tips on using ICAfectin®442 siRNA transfection to perform DNA transfection Mammalian cells - Immortalized cell lines HeLa

Get tips on using Dulbecco’s Modified Eagle’s Medium (DMEM) (1X),liquid to perform Mammalian cell culture media HSG cells

Get tips on using FITC Annexin V Apoptosis Detection Kit I to perform Apoptosis assay cell type - B-cells

Get tips on using FITC Annexin V Apoptosis Detection Kit I to perform Apoptosis assay cell type - HeLa cells

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.